Background: Chewing of sticks was a method of cleaning teeth in ancient India and is still followed by many people in the rural parts. People of different religions also hold high value for these plants that are of high medicinal value and have antimicrobial effects. Objectives: We previously in a study tried to evaluate the antibacterial efficacy of plant extracts from neem, miswak, liquorice, babul, pine, and black cumin against Lactobacillus acidophilus and Streptococcus mutans and have seen few positive results. In this study, we aim to use these extracts to determine if they show antifungal efficacy. Materials and Methods: Chewing sticks of neem, babul, miswak, liquorice, and the leaves of pine and seeds of black cumin were collected from plants sources and sun-dried after which they were ground into coarse powder. Sterile aqueous solutions of 5%, 10%, and 20% were prepared. Aqueous extraction and cold infusion were two methodologies we used. The filtrate was inoculated onto Sabouraud dextrose agar plate with chloramphenicol containing Candida and incubated at 37°C for 48 h. Results: Pine and black cumin showed good antifungal efficacy, on increasing the concentration the extracts of miswak and liquorice also showed a better antifungal efficacy. The combination of these plant extracts had better efficacy than individual extracts of babul and pine. The extracts obtained from cold infusion method almost showed equal antifungal efficacy in comparison to the standard chlorhexidine used. Conclusion: These plants extracts previously having showed antibacterial efficacy and good antifungal effects can be considered for preparations of commercial antiplaque agents.

The oral cavity is a region constantly thriving with thousands of microbial agents. Most of these agents are commensals to the human being and do not cause disease. Candida albicans is one such organism that is a commensal but in poor oral hygiene and in the immunocompromised patients thrives to cause oral thrush. Consistent increase in the cases of oral candidosis with the increase in number of AIDS patients and the HIV epidemic. There is also significant role of candida in many oral and precancerous lesions,[2] the nitrosamines of candida thus making it a potent carcinogen. Putative virulence factors of Candida include the ability to adhere to host surfaces, produce filamentous growth forms, and release hydrolytic enzymes capable of inducing damage to host cells.[3] The plant extracts having antifungal efficacy could be a significant alternative to chemical mouthwashes available.

Aims and objectives

In this study, we aim to assess by doing an in vitro study using plant extracts of indigenous plants widely available in India such as miswak (Salvadora persica), neem (Azadirachta indica), liquorice (Glycyrrhiza glabra), babul (Vachellia nilotica), black cumin (Nigella sativa), and pine (Pinus) and assess whether that these plant extracts that in our previous study which showed significant antibacterial efficacy against Lactobacillus and Streptococcus also have antifungal efficacy and to determine their probable synergistic effects.[1]

Materials and Methods

Material collection

Twigs of neem (Azadirachta indica), miswak (Salvadora persica), liquorice (Glycyrrhiza glabra), babul (Vachellia nilotica) chewing sticks, and pine leaves (Pinus) were collected and black cumin seeds (Nigella sativa) were brought from an open market from Chennai. The experiments were carried out at Department of Microbiology, SRM Dental College, Chennai. Wild strains of Candida albicans were used. The quality control of sterility media and organism was verified to satisfaction. The positive control used for the study was chlorhexidine as it is widely used in mouthwashes. Negative control was saline.

Preparation of extracts

The same methodologies of extractions we had applied for the previous study was used in this study.[1]

Water extraction method

The sticks of miswak, neem, babul, and liquorice; seeds of black cumin and leaves of pine were sun-dried, ground into coarse powder, and weighed into 5 g, 10 g, and 50 g amounts of each powder. 1 g of each powder, 2 g of each powder, and 8 g of each powder was weighed and added to obtain a combined weights of 6 g, 12 g, and 48 g, respectively. These were added to 100 ml of deionized distilled water. After soaking for 48 h at 4°C, the water was filtered using Whatman filter. The filtrate was inoculated on wells containing specific Candida albicans.

Cold infusion method

The sticks of miswak, neem, babul, and liquorice; seeds of black cumin and leaves of pine were sun-dried, ground into coarse powder, and weighed 2 g each and added to 10 ml of ethanol. For combination 6 g of each powder was weighed and mixed to obtain a combined weight of 36 g, and this was added to 180 ml of ethanol. These were hand shaken every 2 h for 15 min for 48 h. The filtrate was filtered using Whatman filter paper and allowed to dry completely. The dried extracts were then mixed with required quantity of dimethyl sulfoxide/dimethylformamide to obtain the working concentration of the extract (100 mg/ml).

Results

[Table 1] shows antifungal efficacy, miswak which was strong antibacterial showed poor antifungal effect. In 5 mg aqueous extract, pine and black cumin showed good efficacy comparative to others, whereas with the increase in the concentration of extract, miswak, liquorice, black cumin, and neem showed better antifungal efficacy. In 50 mg extract, black cumin continued to show steady antifungal effect. Pine that showed considerable antifungal efficacy in aqueous extract also has a good zone of inhibition with respect to the cold infusion method better than the other ethanolic extracts. On increasing concentrations, the ethanolic extracts were difficult to obtain as they obtained a clay-like consistency.

[Table 2] shows the antifungal efficacy of all six herbal products combined together (miswak, babul, black cumin, neem, pine, and liquorice). It was seen that at 1 mg of each product combined together (6 mg concentration of the combined plant material extract), the efficacy was comparatively lower, but on increasing the concentration to 2 mg of each product (12 mg the combined plant material), the zone of inhibition is as effective and equivalent to chlorhexidine. On further increasing the dose to 8 mg of each product (48 g of the combined plant material), it was observed that a dough-like consistence as obtained which makes it difficult to obtain an aqueous extract.

Pine and black cumin proved to be strong antifungals as comparative to that of the control in aqueous extract. The aqueous extract of black cumin but failed to show significant effect in ethanolic extract. The combined extract in 12 g concentrate was as effective as the control in the aqueous extraction method, whereas the combined ethanolic extract failed to show antifungal effect. On subjecting to statistical analysis [Table 3], it was further concluded that comparisons across 5% and 10% concentration of aqueous extract yielded significant positive results (t = 0.006, df = 7, highly statistically significant), proving that the antifungal efficacy of the extract increased with increasing concentration; however, we failed to find significant results at 50% concentrations as most of the extracts were not obtained. It was also observed the statistical difference across the two methods aqueous extraction, and cold infusion was also significant (t = 0.02, df = 7, statistically significant) proving that cold infusion method is comparatively superior when compared with aqueous extraction method.

The extracts obtained from aqueous extraction and cold infusion methods which previously showed significant antimicrobial efficacy were now tested against Candida albicans. All the plant extracts that were taken up showed some level of antibacterial effects in our initial study.[1] In other earlier study conducted, it was indicated that the compound extracted from Salvadora persica can contain compounds with therapeutic potential against Candida strains.[4] Acacia nilotica, commonly called babul, has been used as an oral hygiene adjuvant from ages, but still, studies are lacking on the antifungal property of babul on the most common oral fungus.[5] Previous studies showed that the compounds of liquorice had an inhibitory effect on Candida albicans biofilm formation.[6] Solvent derived extracts from Azadirachta indica showed good anticandidal effect against Candida albicans in a previous conducted study.[7] De et al. and Khan et al. reported that the extract from Nigella sativa seeds had antifungal activity against Aspergillus parasiticus, Candida albicans, and Saccharomyces cerevisiae, respectively.[8],[9] Pine did not have any studies done before to evaluate for its antimicrobial effects. In the present study, pine and black cumin had the highest antifungal effect compared to the other extracts when individually assessed for efficacy but was still lower than the zone of inhibition of chlorhexidine used as control. This is the first of its kind study to check antifungal efficacy of pine against Candida albicans and extracts from pine leaves showed significant antifungal efficacy in increased concentrations. The combination of these plant extracts at a higher concentration showed almost equal antifungal efficacy comparable to the control suggestive of strong synergism; however, further studies and clinical evaluation will be necessary for probable therapeutic usage.

Conclusion

In this study out of the six different plant extracts pine and black cumin has shown good antifungal efficacy and on increasing their concentration the extracts of miswak and liquorice also showed a better antifungal efficacy. The combination of these plant extracts had better efficacy than individual extracts of babul and pine. Whereas the extracts obtained from cold infusion method almost showed equal antifungal efficacy in comparison to the standard chlorhexidine used. These plants extracts can be considered for preparations of antiplaque agents as they have both antibacterial and antifungal activity and thus can be an effective alternative to commercial chemical antiplaque agents. Various other methods of preparation of extracts can also be experimented with, which can be a future scope for this study to evaluate if other methods can give better efficacy. As our study was conducted in vitro with the plant extracts, the action of these extracts with the microbes in the oral cavity in vivo is not clear. Further studies studying the prevalence of diseases like periodontitis, dental caries among subjects who used these plant extracts in the form chewing sticks could enhance our knowledge about their use.

Sulthana S, Swapna IS, Mungara MS, Manipal S, Prabu D, Rajmohan M, et al. Comparitive evaluation of the antimicrobial efficacy of six different plant extracts and their synergistic effect against streptococcus mutans and lactobacillus: An in vitro study. International Journal of Current Research 2017;9(06):53239-41.